The use of therapeutic products obtained from human plasma, such as clotting factors, with the purpose of use in therapy, especially in the case of hereditary bleeding troubles, such as haemophilia, can be greatly jeopardized due the presence of viruses in blood products, which are a high risk to the haemophiliac patient. In spite of the rigorous selection of individual donors, there is a continuous risk of transmission of various viruses, especially of hepatitis and AIDS, and of yet unknown viruses, which may reveal themselves to be transmissible by blood products.
Therefore, the virus transmission should be avoided by means of suitable treatments of the different purified fractions obtained from donor plasma and intended to therapeutic use. On this account, various virus inactivation and elimination methods applied to various protein fractions obtained from blood plasma, are well known. For instance, solvent-detergent treatments, ultrafiltration and nanofiltration, pasteurisation or extended heating, can be mentioned. In the case of extended heat treatment, this can usually be applied only to plasma protein fractions subjected to a preliminary freeze-drying, while requiring heating temperatures of at least 70° C. in a laps of time between 50 and 100 hours, in order to obtain an optimal virus inactivation. However, under such severe heat treatment conditions, the sensitive and heat-instable plasma proteins undergo degradations, which yield important decrease of their biological functions.
In order to find a solution to this drawback, protecting excipients and stabilizers for plasma proteins are beforehand added to liquid compositions of proteins prior to freeze-drying, in order to fulfill a joint double aim. The first aim meets the need to stabilize, on one hand, the considered proteins during the freeze-drying and, on the other hand, the freeze-dried proteins during the storage, and the second aim corresponds to the need to protect the freeze-dried proteins during the heat treatment of virus inactivation.
A method of heating of protein fractions from freeze-dried plasma, Factor VIII or fibrinogen, disclosed in the patent EP 0 094 611, consists of heating the dry product at a temperature of 60° C. for from 72 to 96 hours. No specific composition of stabilizing excipients during the heat treatment is mentioned in this patent.
The canadian patent 1 260 389 mentions the incorporation of excipients, such as non-polar anions with molecular weights higher than 80, especially sugars, reducing sugars and amino acids, into liquid compositions of plasma proteins prior to freeze-drying, in order to stabilize these proteins against the dry heating for of about 72 hours at 68° C. However, the association of reducing sugars with amino acids leads to Maillard compounds, the properties of which do not account for the safety of the treated proteins (activity, immunogenicity, allergies, etc.). This treatment has to be carried out in vacuum or in an inert atmosphere.
Most of the stabilizing excipients may prove to be protective of plasma proteins fractions during the dry heat treatment thereof, at temperatures ranging from of 60° C. to 68° C. for of 30 to 96 hours (P. Thomas, British Journal of Haematology, 70, 1998, 393-395 and J. A. Levy et al., The Lancet, Jun. 22, 1985, 1456-1457). It was shown, however, that, in spite of a diminution of the viral titer after such a treatment under these conditions, infections, such as HIV, HBV, HBC and parvovirus B19 could, nevertheless, been transmitted (P. Thomas, supra). In view of their effective elimination, it was suggested to heat the freeze-dried plasma proteins fractions to higher temperatures. So S. J. Skidmore et al. (Journal of Medical Virology, 30, 1990, 50-52) have shown that a heat treatment of freeze-dried Factor VIII concentrates at a temperature of 80° C. for 72 hours avoids the transmission of HCV non-A and non-B virus. So the U.S. Pat. No. 5,831,027 discloses a process of heat treatment of a freeze-dried protein obtained from the cryoprecipitate of blood plasma, the fibrinogen, at a temperature of 80° C. for 72 hours, which allows to obtain a fibrinogen free of possible viruses, such as HBV, HBC or parvovirus B19. The stabilizing excipients, added to protect the fibrinogen composition during both the freeze-drying and the heat treatment of virus inactivation, include sucrose and/or an amino acid (arginine), Tris buffer and sodium citrate. L. Wilkelman et al. (Virus Inactivation in Plasma Products, Curr. Stud Hematol Blood Transfus., Basel, Karger, 1989, no 56, 55-69) also show the need of addition of excipients to Factor VIII prior to freeze-drying and to heat treatment of virus inactivation at 80° C. for 72 hours. The described excipients are: NaCl, sodium citrate, Tris, CaCl2 and sucrose.
Besides, as the different proteins obtained by plasma fractionation, having been subjected to freeze-drying and to heat treatment of virus inactivation, require a reconstitution in a suitable medium prior to their clinical use, this should be easy to carry out in a relatively short laps of time according to the requirements recommended by the European Pharmacopoeia. In this respect, studies of the heat stability of Factor VIII in a freeze-dried cryoprecipitate (J. Margolis et al., The Lancet, Dec. 8, 1984, 1345) containing, prior to freeze-drying, Synthamin 17% (Travenol Laboratories Ltd.), a mixture of natural amino acids suited to intravenous administration, have shown that a heat treatment at 80° C. for 16 hours leads not only to such an extent of degradation of Factor VIII that its activity equals to zero, but also to an impossibility of a redissolution of the cryoprecipitate after the mentioned steps. The U.S. Pat. No. 5,399,670 discloses a process facilitating the solubilization or the reconstitution of compositions of freeze-dried Factor VIII complex in purified water for injections, including a step of addition of arginine to a Factor VIII solution prior to freeze-drying thereof. This patent does not mention a heat treatment of virus inactivation. An addition of histidine and albumin can also be provided. The stabilizing excipients, mentioned in the previously cited U.S. Pat. No. 5,831,027, are also aimed to favour the dissolution of freeze-dried fibrinogen in pure water, prior to therapeutic use.
Nevertheless, the choice of a stabilizing formulation is governed by the specificity of the plasma proteins. Thus, in reference to a paper by N. Heimburger et al. (Virus Inactivation in Plasma Products., Curr Stud Hematol Blood Transfus., Basel, Karger, 1989, No. 56, 23-33), it is usually considered that a specific stabilizing formulation can be suited to only one protein fraction containing the given active proteins of interest. A further difficulty appears in the case where more complex protein fractions are considered, especially those, where all coagulation and haemostasis proteins obtained from plasma fractionation are taken into account. Moreover, carbohydrates, especially sucrose, may effectively be used as excipients for the stabilization and the redissolution of plasma proteins fractions, when the considered fractions are intended to be freeze-dried, then subjected to dry heat treatment, although their effect slows down the virus inactivation (N. Heimburger et al., supra). Therefore, the protein fractions treated in that way may not be completely virus-free and their use in clinics is thus restricted. Furthermore, some carbohydrates, such as maltose or sucrose, cannot be safely used with subjects suffering from kidney deficiency and/or diabetes.